The overall objective of this proposal is to continue to test our hypothesis that activation of Raf-1 kinase requires ordered and cooperative actions of multiple factors including kinases and scaffold proteins. Raf family members are major downstream effectors of Ras and key enzymes in the regulation of cell metabolism, proliferation, differentiation and apoptosis. The Raf kinases are also important factors in neoplasia. Thus, activating mutations of the Ras gene are found in approximately 30% of human cancers and many non-nuclear oncogenes exploit the Ras/Raf/MEK/Erk pathway to execute their oncogenic programs. Previous studies by others and us have demonstrated that different inputs lead to the phosphorylation of S338, which is necessary, but not sufficient for Raf-1 activation. Our preliminary data suggest that S338 is autophosphorylated in response to growth factors (e.g., EGF or TPA), and that it is phosphorylated by Pak in response to microtubule depolymerizing agents such as nocodazole. We have identified several novel phosphorylation sites (e.g. S357) on Raf-1 that appear to participate in its regulation. In addition, we have found that Raf-1 is constitutively activated in the HCT116 colon cancer cell that contains an activating mutation of KiRas. We have also found that suppression of KSR expression by RNAi results in inhibition of Raf/MEK interaction and MEK activation, leading to DNA fragmentation. Finally, our preliminary results indicate that the HCT116 cell extract contains component(s) that allows Raf-1 activation in vitro. Based on these findings, we are proposing studies with the following specific aims: (1) to examine further how S338 is phosphorylated under different conditions and its relationship with other phosphorylation events; (2) to examine the mechanism(s) responsible for the phosphorylation of S357 and to determine the impact of this phosphorylation on Raf function and vRas-mediated transformation (malignancy); and (3) to elucidate the mechanism(s) by which Raf-1 is regulated by KSR in HCT116 cells and to use these cells as a tool to identify novel upstream regulators of Raf-1. These studies will provide new information as to how Raf is activated and how its activation affects cellular functions. They will also serve as a framework for evaluating the role of the Raf family in human malignancy.